High Level Of Protein Synthesis Research Articles

The cytidine deaminase APOBEC3A regulates nucleolar function to promote cell growth and ribosome biogenesis.

Cancer initiates as a consequence of genomic mutations and its subsequent progression relies in part on increased production of ribosomes to maintain high levels of protein synthesis for unchecked cell growth. Recently, cytidine deaminases have been uncovered as sources of mutagenesis in cancer. In an attempt to form a connection between these 2 cancer driving processes, we interrogated the cytidine deaminase family of proteins for potential roles in human ribosome biogenesis. We identified and validated APOBEC3A and APOBEC4 as novel ribosome biogenesis factors through our laboratory's established screening platform for the discovery of regulators of nucleolar function in MCF10A cells. Through siRNA depletion experiments, we highlight APOBEC3A's requirement in making ribosomes and specific role within the processing and maturation steps that form the large subunit 5.8S and 28S ribosomal (r)RNAs. We demonstrate that a subset of APOBEC3A resides within the nucleolus and associates with critical ribosome biogenesis factors. Mechanistic insight was revealed by transient overexpression of both wild-type and a catalytically dead mutated APOBEC3A, which both increase cell growth and protein synthesis. Through an innovative nuclear RNA sequencing methodology, we identify only modest predicted APOBEC3A C-to-U target sites on the pre-rRNA and pre-mRNAs. Our work reveals a potential direct role for APOBEC3A in ribosome biogenesis likely independent of its editing function. More broadly, we found an additional function of APOBEC3A in cancer pathology through its function in ribosome biogenesis, expanding its relevance as a target for cancer therapeutics.

Comparative transcriptomic analysis delineates adaptation strategies of Rana kukunoris toward cold stress on the Qinghai-Tibet Plateau

BackgroundCold hardiness is fundamental for amphibians to survive during the extremely cold winter on the Qinghai-Tibet plateau. Exploring the gene regulation mechanism of freezing-tolerant Rana kukunoris could help us to understand how the frogs survive in winter.ResultsTranscriptome of liver and muscle of R. kukunoris collected in hibernation and spring were assisted by single molecule real-time (SMRT) sequencing technology. A total of 10,062 unigenes of R. kukunoris were obtained, and 9,924 coding sequences (CDS) were successfully annotated. Our examination of the mRNA response to whole body freezing and recover in the frogs revealed key genes concerning underlying antifreeze proteins and cryoprotectants (glucose and urea). Functional pathway analyses revealed differential regulated pathways of ribosome, energy supply, and protein metabolism which displayed a freeze-induced response and damage recover. Genes related to energy supply in the muscle of winter frogs were up-regulated compared with the muscle of spring frogs. The liver of hibernating frogs maintained modest levels of protein synthesis in the winter. In contrast, the liver underwent intensive high levels of protein synthesis and lipid catabolism to produce substantial quantity of fresh proteins and energy in spring. Differences between hibernation and spring were smaller than that between tissues, yet the physiological traits of hibernation were nevertheless passed down to active state in spring.ConclusionsBased on our comparative transcriptomic analyses, we revealed the likely adaptive mechanisms of R. kukunoris. Ultimately, our study expands genetic resources for the freezing-tolerant frogs.

Abstract 450: GCN2 protects cancer cells from hypertranslation and metabolic crisis

Abstract It is widely assumed that cancer cells rely on high levels of protein synthesis to support growth and proliferation. However, protein synthesis, including ribosome biogenesis, is a highly resource- and energy-demanding process that needs to be coordinated with protein degradation and metabolic programs. GCN2 is an evolutionarily conserved kinase that is primarily activated by uncharged tRNAs when amino acid levels drop and activates the Integrated Stress Response (ISR), which encompasses a global reduction in translation and increased amino acid uptake and generation. GCN2 has been shown to support cancer cell survival under conditions of nutrient scarcity. Here, we used an integrated systems biology approach to delineate the multifaceted role of GCN2 in cancer cells that do not suffer from nutrient shortages. We carried out a broad array of multiomic analyses (RNA-seq, TMT-labelling based proteomics, Ribo-seq, LC-MS metabolomics) of melanoma cells in which GCN2 was inhibited (GCN2iB) or genetically depleted (constitutive and inducible shRNA) in cell culture and in vivo in murine xenografts, supplemented by puromycinylation-based quantification of global protein synthesis. GCN2 inhibition in A375 cells triggered a transcriptome and proteome response that was dominated by the induction of processes linked to protein and ribosome synthesis while suppressing metabolic pathways such as glycolysis and the proteasome. We confirmed these observations in vivo via RNA-seq of tumor samples from mice xenografted with A375 cells in which GCN2 was knocked down by induced shRNA expression and from xenografted mice that were treated orally with GCN2iB. Ribo-seq further demonstrated that, upon GCN2 inhibition, A375 cells expand their translational machinery. Notably, we identified 50 ribosomal genes that were translated at higher rate in GCN2-inhibited cells and whose expression was regulated exclusively at the translational level. GCN2 inhibition/depletion also resulted in an approximately 1.8-fold increase in global protein synthesis and altered the druggable proteome. Moreover, metabolite profiling of GCN2iB-treated cells revealed a loss of metabolic homeostasis including substantial alterations in glycolysis and both amino acid and lipid metabolism. In addition, we observed a significant decrease in ATP levels despite increased translation of mitochondrial ETC complex proteins. Proteomic and translatomic analyses revealed a failure of cells to increase proteasomal subunits and to maintain the production of ECM proteins. Last, but not least, we did not observe a role of GCN2 in preventing or resolving ribosome collisions. Taken together, our findings reveal GCN2 as a master regulator of translation in cancer cells that do not suffer from nutrient scarcity that keeps protein synthesis in check to maintain metabolic homeostasis. Citation Format: Monica Roman-Trufero, Istvan T. Kleijn, Kevin Blighe, Paula Saavedra-Garcia, Jinglin Zhou, Abigail Gaffar, Marilena Christoforou, Mariia Yuneva, Audrey M. Michel, Glenn R. Masson, Vahid Shahrezaei, Holger W. Auner. GCN2 protects cancer cells from hypertranslation and metabolic crisis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 450.

Abstract 6271: Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer

Abstract Prostate cancer (PC) stands as the most frequently diagnosed and second leading cause of cancer death among American men. PC mortality is predominantly attributed to castration-resistant prostate cancer (CRPC), often emerging within 2-3 years following the initiation of androgen deprivation therapy. This resistance primarily arises from sustained androgen receptor (AR) signaling, ultimately resulting in a compromised response to AR-directed therapies. AR splice variant expression is a key contributor to the persistent AR signaling in CRPC. AR-variant 7 (AR-V7) is the most clinically prevalent variant, expressed in about 75% of patients with CRPC and confers resistance to standard of care (SoC) treatments. AR-V7 differs from the full-length AR (AR-fl) because it lacks the ligand binding and hinge domains, rendering it resistant to SoC treatments. Currently, there are no selective inhibitors for AR-V7. Therefore, we sought to identify unique biological features of AR-V7, distinct from AR-fl, to exploit therapeutic options for targeting AR-V7. Previous studies have demonstrated high sequence homology, largely overlapping cistromes, and gene transactivation profiles between AR-V7 and AR-FL. Our published mechanistic studies revealed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl, allowing for constitutive import and activation of target genes in the nucleus. Herein, we report an additional novel subnuclear phenotype of AR-V7 in LNCAP95 and 22RV1 cells modeling CRPC. Staining with AR-V7- and AR-fl- specific antibodies revealed that unlike AR-fl, AR-V7 exhibited prominent nucleolar localization. To validate AR-V7 nucleolar localization, CRPC cells stained with fibrillarin (nucleolar marker), and subsequent fluorescence intensity analysis revealed that AR-V7 was predominantly localized in fibrillarin positive nucleoli, whereas AR-fl protein was solely localized in the nucleoplasm. This suggests a novel functional role of AR-V7 in the nucleus. To further confirm AR-V7 nucleolar localization, Actinomycin D (ActD) was used as a tool compound to disrupt nucleolar integrity. Treatment with ActD for 4 hours in CRPC cells significantly reduced the area of fibrillarin positive nucleoli and prompted AR-V7 re-distribution out of the nucleoli and into the nucleoplasm. Ongoing efforts to further investigate AR-V7 nucleolar function in CRPC cells include mutating the predicted nucleolar localization signal located in AR-V7 and identifying nucleolar-interacting proteins through proximity-labeling assays. In conclusion, we unveiled a novel nuclear compartmentalization of AR-V7 within the nucleolus, the primary hub for ribosomal biogenesis. These observations suggest that AR-V7 could regulate the high levels of protein synthesis occurring in CRPC cells, creating a window of a potential therapeutic opportunity for selective inhibition. Citation Format: Michelle K. Naidoo, Paraskevi Giannakakou. Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6271.

СОВРЕМЕННЫЕ МИКРОБИОЛОГИЧЕСКИЕ ПОДХОДЫ К ПОЛУЧЕНИЮ ШТАММА ХЛЕБОПЕКАРНЫХ ДРОЖЖЕЙ, ПЕРСПЕКТИВНОГО ДЛЯ СОЗДАНИЯ ОБОГАЩЕННЫХ ПИЩЕВЫХ ИНГРЕДИЕНТОВ

The aim of the study is to use modern microbiological approaches to obtain a new strain of baker's yeast. The objects of the study were strains of baker's yeast from the Laboratory of Baker's Yeast Biotechnology of the Institute of Food Biotechnology. Selection and screening of the yeast strain were carried out by clonal plating of the original strain cells on agar media with glucose-asparagine medium (8 % DM) and with malt wort (12 and 18 % DM). The content of crude protein in the yeast biomass was determined by the Kjeldahl method on an automatic BEGER unit (Slovenia). Biomass accumulation was determined by the gravimetric method after cultivation, separating the solid and liquid fractions by centrifugation. Maltase, zymase and proteolytic activities were determined according to known methods. Ergosterol content – spectrophotometric method based on extraction with ethanol and sulfuric acid with triterpene compounds, at a wavelength of λ = 328 nm. The biomass (solid fraction) was separated from the filtrate by centrifugation in a laboratory centrifuge OPM-16 for 15 min at a rotor speed of 6000 rpm. Comparative studies were conducted to select the most productive strain of baker's yeast in terms of biochemical parameters. Comparative characteristics of yeast strains allowed us to select the most promising one – Saccharomyces cerevisiae Y-581, characterized by the ability to a higher level of protein synthesis, maltase, zymase activities, as well as ergosterol and osmosensitivity indicators. A two-stage selection and screening for the above indicators were carried out with the selected strain. As a result of microbiological approaches relative to the original strain S. cerevisiae Y-581, a population of Saccharomyces cerevisiae 581-GA-21c was isolated, characterized by a higher biosynthetic capacity not only in relation to ergosterol and protein, but also to the synthesis of enzymes, especially proteolytic ones. The isolated strain had a higher permeability of the cell membrane, as evidenced by its osmosensitivity indicators. The new strain also had distinctive features in morphological characteristics. This strain will be used in the field of obtaining enriched ingre¬dients for the development of new types of food products.

Anterior Pituitary Transcriptomics Following a High-Fat Diet: Impact of Oxidative Stress on Cell Metabolism.

Anterior pituitary cell function requires a high level of protein synthesis and secretion which depend heavily on mitochondrial adenosine triphosphate production and functional endoplasmic reticula. Obesity adds stress to tissues, requiring them to adapt to inflammation and oxidative stress, and adding to their allostatic load. We hypothesized that pituitary function is vulnerable to the stress of obesity. Here, we utilized a 10- to 15-week high-fat diet (HFD, 60%) in a thermoneutral environment to promote obesity, testing both male and female FVB.129P mice. We quantified serum hormones and cytokines, characterized the metabolic phenotype, and defined changes in the pituitary transcriptome using single-cell RNA-sequencing analysis. Weight gain was significant by 3 weeks in HFD mice, and by 10 weeks all HFD groups had gained 20 g. HFD females (15 weeks) had increased energy expenditure and decreased activity. All HFD groups showed increases in serum leptin and decreases in adiponectin. HFD caused increased inflammatory markers: interleukin-6, resistin, monocyte chemoattractant protein-1, and tumor necrosis factorα. HFD males and females also had increased insulin and increased TSH, and HFD females had decreased serum prolactin and growth hormone pulse amplitude. Pituitary single-cell transcriptomics revealed modest or no changes in pituitary cell gene expression from HFD males after 10 or 15 weeks or from HFD females after 10 weeks. However, HFD females (15 weeks) showed significant numbers of differentially expressed genes in lactotropes and pituitary stem cells. Collectively, these studies reveal that pituitary cells from males appear to be more resilient to the oxidative stress of obesity than females and identify the most vulnerable pituitary cell populations in females.

Translational Control of Metabolism and Cell Cycle Progression in Hepatocellular Carcinoma.

The liver is a metabolic hub characterized by high levels of protein synthesis. Eukaryotic initiation factors, eIFs, control the first phase of translation, initiation. Initiation factors are essential for tumor progression and, since they regulate the translation of specific mRNAs downstream of oncogenic signaling cascades, may be druggable. In this review, we address the issue of whether the massive translational machinery of liver cells contributes to liver pathology and to the progression of hepatocellular carcinoma (HCC); it represents a valuable biomarker and druggable target. First, we observe that the common markers of HCC cells, such as phosphorylated ribosomal protein S6, belong to the ribosomal and translational apparatus. This fact is in agreement with observations that demonstrate a huge amplification of the ribosomal machinery during the progression to HCC. Some translation factors, such as eIF4E and eIF6, are then harnessed by oncogenic signaling. In particular, the action of eIF4E and eIF6 is particularly important in HCC when driven by fatty liver pathologies. Indeed, both eIF4E and eIF6 amplify at the translational level the production and accumulation of fatty acids. As it is evident that abnormal levels of these factors drive cancer, we discuss their therapeutic value.

Control of immune cell function by the unfolded protein response.

Initiating and maintaining optimal immune responses requires high levels of protein synthesis, folding, modification and trafficking in leukocytes, which are processes orchestrated by the endoplasmic reticulum. Importantly, diverse extracellular and intracellular conditions can compromise the protein-handling capacity of this organelle, inducing a state of 'endoplasmic reticulum stress' that activates the unfolded protein response (UPR). Emerging evidence shows that physiological or pathological activation of the UPR can have effects on immune cell survival, metabolism, function and fate. In this Review, we discuss the canonical role of the adaptive UPR in immune cells and how dysregulation of this pathway in leukocytes contributes to diverse pathologies such as cancer, autoimmunity and metabolic disorders. Furthermore, we provide an overview as to how pharmacological approaches that modulate the UPR could be harnessed to control or activate immune cell function in disease.

PS-BPB07-6: NOVEL HUMORAL AND NEURONAL PATHWAYS FOR DIRECT BLOOD PRESSURE CONTROL BY MACULA DENSA CELLS

Objective: According to traditional physiological knowledge, macula densa (MD) cells of the juxtaglomerular apparatus (JGA) in the kidney maintain body fluid homeostasis and blood pressure (BP) indirectly by controlling renal and glomerular hemodynamics and renin secretion based on local sensory inputs. However, recently identified microanatomical (long, axon-like basal cell processes, secretory vesicles) and functional (high level of protein synthesis) features of MD cells suggest they perform non-traditional functions. The present study addressed the hypothesis that MD cells can directly and acutely control distant cardiovascular organ functions and blood pressure via novel secreted hormones and communications with renal nerves. Design and method: A comprehensive research approach used transgenic mouse models (MD-GFP, MD-Ai27 mice that selectively express the depolarizing channel rhodopsin ChR2 in MD cells), intravital multiphoton imaging, systemic BP monitoring via the cannulated carotid artery, whole mount kidney imaging and 3D analysis, and single-cell transcriptomic analysis. Results: Bulk and single cell MD transcriptome analysis identified the high expression of secreted angiogenic and vasoactive factors (Pappa2, Vash2, Cyr61, Aard, Pamr1) and synaptic proteins (Unc5d, Tenm2, Hpcal4, Nsg2, Syp, Syt5, Grin2c, Gabrr2, Begain) with their altered kidney and plasma levels in disease and MD gain and loss-of-function conditions. Immunohistochemistry and 3D tissue volume rendering of optically cleared whole-mount MD-GFP kidneys identified the high MD expression of synaptophysin and the close anatomical association between MD cell basal processes and tyrosin-hydroxylase (TH)+ or calcitonin gene-related peptide (CGRP)+ sympathetic and sensory nerve endings. Optogenetic MD stimulation was induced by blue light (470 nm) exposure of the whole left kidney of MD-Ai27 mice, which maneuver acutely and reversibly increased systemic BP by 22 ± 5mmHg (P < 0.05, n = 5). Conclusions: MD cells secrete hormones that target the vasculature and form synapses with sympathetic and sensory renal nerves. These novel MD and JGA pathways may be involved in direct BP control and represent exciting future therapeutic opportunities for renal and cardiovascular disease and hypertension.

Protein metabolism and immune response levels in cuttlefish (Sepia pharaonis) and the effects of inking stress on development

Inking is a defensive behavior that protects cephalopods from predator attack, a process that is accomplished by generating jet propulsion from powerful contractions of the mantle muscle. However, most studies have focused on the “defensive effects” of the ink, with the result that there is limited information concerning the effects of inking on the animals themselves. On the basis of our previous research, we integrated information from different tissues after continuous ink release to develop a better understanding of the physiological stress resulting from inking in the cuttlefish (Sepia pharaonis). The results showed that throughout the recovery period (60 days) of the inking treatment, the observed declines in growth performance due to decreased protein synthesis and increased energy metabolism levels were supported by a decrease in mantle muscle glycogen concentration (P < 0.01), increased hexokinase (HK) and pyruvate kinase (PK) specifical activities (P < 0.001), up-regulated expression of arginine kinase (AK) and octopine dehydrogenase (ODH) (P < 0.001), and up-regulated immune superoxide dismutase (SOD) and heat shock protein 70 (HSP70) in the digestive gland (P < 0.001). Muscle principal component analysis (PCA) revealed that amino acids (lysine, tyrosinase, arginine, leucine, isoleucine, and aspartate) were involved in protein turnover after the ink release, and essential fatty acids such as oleic acid (18:1n-9), linoleic acid (18:2n-6), arachidonic acid (ARA, 20:4n-6), and docosahexaenoic acid (DHA, 22:6n-3) were disturbed by inking stress. Moreover, the inducible HSP70 in the digestive gland was sensitively up-regulated even after the 60-day recovery, and thus the transcription level of induced HSP70 in cuttlefish may be used as an indicator of physiological stress. In conclusion, a high growth rate requires a high level of protein synthesis, and it appears that considerable ink release has a significant impact on cuttlefish (S. pharaonis) physiological stress. The transcription level of HSP70 may be used as a stress indicator

MTORC1 regulates high levels of protein synthesis in retinal ganglion cells of adult mice

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.

Size and Age Characteristics and Phenotypic Peculiarities of Somatic Growth of the Black Sea Mollusk Flexopecten glaber ponticus (Bivalvia, Pectinidae)

This paper presents a preliminary analysis of the morphological, physiological, and biochemical parameters of somatic growth in the Black Sea scallop, Flexopecten glaber ponticus Bucguoy, Dautzenberg et Dollfus 1889, from a coastal site near Sevastopol, Crimea. The species is one of the few marine Black Sea mollusks listed in the Red Book of Sevastopol and Crimea with the status “Diminishing in numbers.” The peculiarities of soft tissue somatic growth have been studied in three size groups differing in linear diameter: 13–17, 21–24, and 25–30 mm. An analysis of the total RNA in tissue homogenates has shown that the rate of protein synthesis in the first and second groups (under-yearlings) is 1.2–1.5 times higher compared to adults. The population reveals seven color morphs (phenotypes). Three phenotypes (beige, violet, and gray-and-brown) are characterized by high-level protein synthesis. Mollusks of a mixed “multi” phenotype have the lowest level of tissue biosynthesis, usually being 2.5 times lower than that for other morphs. The results obtained indicate that the growth processes are linked to the color of the mollusk shell. High direct correlations between the weights of the total wet mass and the tissue mass observed in all mollusk groups may be evidence of positive growth allometry.

MDM2's dual mRNA binding domains co-ordinate its oncogenic and tumour suppressor activities.

Cell growth requires a high level of protein synthesis and oncogenic pathways stimulate cell proliferation and ribosome biogenesis. Less is known about how cells respond to dysfunctional mRNA translation and how this feeds back into growth regulatory pathways. The Epstein-Barr virus (EBV)-encoded EBNA1 causes mRNA translation stress in cis that activates PI3Kδ. This leads to the stabilization of MDM2, induces MDM2’s binding to the E2F1 mRNA and promotes E2F1 translation. The MDM2 serine 166 regulates the interaction with the E2F1 mRNA and deletion of MDM2 C-terminal RING domain results in a constitutive E2F1 mRNA binding. Phosphorylation on serine 395 following DNA damage instead regulates p53 mRNA binding to its RING domain and prevents the E2F1 mRNA interaction. The p14Arf tumour suppressor binds MDM2 and in addition to preventing degradation of the p53 protein it also prevents the E2F1 mRNA interaction. The data illustrate how two MDM2 domains selectively bind specific mRNAs in response to cellular conditions to promote, or suppress, cell growth and how p14Arf coordinates MDM2’s activity towards p53 and E2F1. The data also show how EBV via EBNA1-induced mRNA translation stress targets the E2F1 and the MDM2 - p53 pathway.

Obtaining of the recombinant rhIL7-BAPmut fusion protein and its functional characterization

Aim. The purpose was to obtain a functionally active form of the recombinant fusion protein based on the human interleukin-7 (rhIL7) and bacterial alkaline phosphatase (BAPmut). Methods. The DNA sequences encoding rhIL-7 and BAPmut were subcloned into the pET24a(+) plasmid vector containing the 6His-tag sequence for further chromatographic purification of the target protein. The cells of E. coli strain BL21(DE3) were transformed with pET24-rhIL7-BAPmut plasmid vector. Protein synthesis was induced by IPTG and by autoinduction. Bifunctional activity of rhIL7-BAPmut after refolding via dilution is confirmed immunochemically by binding to specific antibodies. Results. RhIL7-BAPmut protein has been designed. It was shown that autoinduction protocol provides a significantly higher level of protein synthesis in E. coli compared with IPTG induction. In vitro method of purification and renaturation of rhIL7-BAPmut from inclusion bodies has been worked out. It has been shown that rhIL7-BAPmut has a specific biological activity after renaturation. Conclusions. The obtained rhIL7-BAPmut protein can be used for screening of immune combinatorial cDNA libraries of immunoglobulins variable genes, that are the sources of specific single chain antibodies. The protein also can be used for qualitative and quantitative analysis of IL-7 receptors.&#x0D; Keywords: IL-7, BAPmut, inclusion bodies, renaturation.

Functionally significant metabolic differences between B and T lymphocyte lineages.

Activation of B and T lymphocytes leads to major remodelling of the metabolic landscape of the cells enabling their post-activation functions. However, naive B and T lymphocytes also show metabolic differences, and the genesis, nature and functional significance of these differences are not yet well understood. Here we show that resting B-cells appeared to have lower energy demands than resting T-cells as they consumed lower levels of glucose and fatty acids and produced less ATP. Resting B-cells are more dependent on OXPHOS, while T-cells show more dependence on aerobic glycolysis. However, despite an apparently higher energy demand, T lineage cells showed lower rates of protein synthesis than equivalent B lineage stages. These metabolic differences between the two lineages were established early during lineage differentiation, and were functionally significant. Higher levels of protein synthesis in B-cells were associated with increased synthesis of MHC class II molecules and other proteins associated with antigen internalization, transport and presentation. The combination of higher energy demand and lower protein synthesis in T-cells was consistent with their higher ATP-dependent motility. Our data provide an integrated perspective of the metabolic differences and their functional implications between the B and T lymphocyte lineages.

Studying the biochemical function of the pea receptor-like kinases sym10, sym37 and k1, required for the legume-rhizobia symbiosis development

Background. Rhizobial Nod factors (NFs), the key regulators of legume-rhizobia symbiosis, act in low concentrations and their biological activity depends on structural features, that suggests the presence of specific receptors in plants. Putative receptors, LysM-receptor-like kinases (LysM-RLKs), were found in model legumes L. japonicus and M. truncatula. However, binding capacity with NFs was only studied for L. japonicus LysM-RLKs. In pea a few candidates for NF receptors like Sym10, Sym37 and K1 were found. Analysis of mutants revealed the importance of these proteins for symbiosis development. However, the biochemical function of these receptors has not been studied.&#x0D; Materials and methods. Sequences encoding extracellular domains (ECDs) of LysM-RLKs Sym10, Sym37, and K1 were cloned in the pRSETa vector. Constructs were introduced in E. coli strain C41 to produce proteins with His6 residues on either the amino or carboxyl terminus. Protein purification was carried out using metal chelate affinity chromatography. The binding capacity with ligand was evaluated using ProteonXPR36 biosensor.&#x0D; Results. To study binding capacity with NFs, we have developed approaches for the synthesis of LysM-RLK Sym10, Sym37 and K1 in soluble form in heterologous system. The high level of protein synthesis was achieved at +28 C using 0,5 mM IPTG in 2-16 hours. Analysis of binding capacity of ECDs with NFs revealed the low affinity using the surface plasmon resonance.&#x0D; Conclusion. The possibility of recombinant receptor synthesis in soluble state in E. coli at high level was demonstrated. Analysis of binding capacity with NFs showed the potential interaction, but with low affinity.

Protein Folding and the Challenges of Maintaining Endoplasmic Reticulum Proteostasis in Idiopathic Pulmonary Fibrosis.

Alveolar epithelial type II (AEII) cells are "professional" secretory cells that synthesize and secrete massive quantities of proteins to produce pulmonary surfactant and maintain airway immune defenses. To facilitate this high level of protein synthesis, AEII cells are equipped with an elaborate endoplasmic reticulum (ER) structure and possess an abundance of the machinery needed to fold, assemble, and secrete proteins. However, conditions that suddenly increase the quantity of new proteins entering the ER or that impede the capacity of the ER to fold proteins can cause misfolded or unfolded proteins to accumulate in the ER lumen, also called ER stress. To minimize this stress, AEII cells adapt by (1) reducing the quantity of proteins entering the ER, (2) increasing the amount of protein-folding machinery, and (3) removing misfolded proteins when they accumulate. Although these adaptive responses, aptly named the unfolded protein response, are usually effective in reducing ER stress, chronic aggregation of misfolded proteins is recognized as a hallmark feature of AEII cells in patients with idiopathic pulmonary fibrosis (IPF). Although mutations in surfactant proteins are linked to the development of ER stress in some rare IPF cases, the mechanisms causing protein misfolding in most cases are unknown. In this article, we review the mechanisms regulating ER proteostasis and highlight specific aspects of protein folding and the unfolded protein response that are most vulnerable to failure. Then, we postulate mechanisms other than genetic mutations that might contribute to protein aggregation in the alveolar epithelium of IPF lung.

Distinct cellular toxicity of two mutant huntingtin mRNA variants due to translation regulation.

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion within exon1 of the HTT gene. The gene generates two mRNA variants that carry either a short or long 3′ untranslated region (3′UTR) while encoding the same protein. It remains unknown whether the two mRNA variants play distinct roles in HD pathogenesis. We found that the long HTT 3′UTR was capable of guiding mRNA to neuronal dendrites, suggesting that some long-form HTT mRNA is transported to dendrites for local protein synthesis. To assay roles of two HTT mRNA variants in cell bodies, we expressed mRNA harboring HTT exon1 containing 23x or 145x CAGs with the short or long 3′UTR. We found that mutant mRNA containing the short 3′UTR produced more protein aggregates and caused more apoptosis in both cultured neurons and HEK293 cells, compared with mutant mRNA containing the long 3′UTR. Although the two 3′UTRs did not affect mRNA stability, we detected higher levels of protein synthesis from mRNA containing the short 3′UTR than from mRNA containing the long 3′UTR. These results indicate that the long HTT 3′UTR suppresses translation. Thus, short-form mutant HTT mRNA will be more efficient in producing toxic protein than its long-form counterpart.

Nucleolin Controls Ribosome Biogenesis through Its RNA-Binding Properties

IntroductionNucleolin (NCL) is a multifunctional, proliferation-associated factor that is overexpressed in many cancers and has already been demonstrated to play a profound role in leukemogenesis (Abdelmohsen and Gorospe, 2012; Shen et al., 2014). This can be linked to an increased synthesis of ribosomal RNA (rRNA). Thus, in leukemic cells, high expression levels of NCL contribute to malignant transformation through the increase of rRNA synthesis, which is required to sustain high levels of protein synthesis. Physiologically, NCL is a highly abundant, nucleolar RNA-binding protein that is implicated in the regulation of polymerase I transcription, post-transcriptional gene regulation, and plays a central role in ribosome biogenesis (Srivastava and Pollard, 1999). To further elucidate the exact role of NCL, this study focused on the characterization of the RNA-binding properties and protein-interactions of NCL in the context of ribosome biogenesis.MethodsIn order to identify transcriptome-wide binding sites and the cellular RNA targets of NCL, PAR-CLIP (photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation) and RIP-Seq (RNA immunoprecipitation sequencing) analyses were carried out in HEK 293 cells. PAR-CLIP is characterized by the incorporation of 4-thiouridine into newly transcribed RNA that causes a T to C conversion in the corresponding cDNA of crosslinked RNA (Hafner et al., 2010). The RNA-binding properties and the interaction of NCL with its identified RNA targets were elucidated by electrophoretic mobility shift assays, isothermal titration calorimetry and size-exclusion chromatography. To further define the role of NCL in ribosome biogenesis and the effect on precursor rRNA levels, siRNA mediated knockdown of NCL was employed followed by RNA sequencing. Furthermore, to characterize the interaction network of NCL on a proteome-wide level, mass-spectrometry was performed.ResultsThis study focuses on the characterization of the RNA-binding properties of NCL and provides the first PAR-CLIP data set of NCL and identifies small nucleolar RNAs (snoRNA) and precursor rRNA as main targets of NCL, both of which were further confirmed by RIP-Seq analysis. Binding sites of NCL were identified in the 5'ETS (external transcribed spacer), after the first cleavage site, in ITS1 and ITS2 (internal transcribed spacer) within the precursor rRNA, indicating that NCL might play a role in the early processing steps of ribosome biogenesis within the nucleolus. Biochemical and structural binding analyses reveal that NCL interacts along the complete precursor region and shows high binding affinity to G/C/U-rich repeat sequences, which is in agreement with the nucleotide composition of the primary rRNA transcript. Moreover, we propose that siRNA mediated knockdown of NCL inhibits polymerase I transcription, which is shown by decreased expression levels of the precursor rRNA transcript. On the proteome-wide level, mass-spectrometry analysis of NCL identified several interaction partners including block of proliferation 1 (BOP1), DEAD-box RNA helicase 18 (DDX18), and 5'-3' exoribonuclease 2 (XRN2) and numerous ribosomal proteins of the small and the large ribosomal subunits including RPS24, RPL11, RPL35A, and RPL36.ConclusionThis study provides evidence that NCL is highly associated with the process of ribosome biogenesis on the proteome- and transcriptome-wide level. Therefore, NCL might serve as a promising biochemical target in the context of increased ribosome biogenesis in cancer. DisclosuresNo relevant conflicts of interest to declare.

Abstract B33: Assessing the role of DGAT activity on lipid homeostasis and cancer cell survival

Abstract Studies on the effects of modulating the lipid composition of cells have found that increases in saturated fatty acid levels can lead to ER stress, activation of the unfolded protein response (UPR) and cell death. These effects may occur through saturation of the phospholipid pool and subsequent deterioration of ER membrane function. Changes in the composition of the ER membrane can be sensed by the two UPR stress sensors IRE1 and PERK and lead to activation of UPR targets. Rapidly proliferating cancer cells frequently exhibit elevated ER stress due to their increased protein translation rate. In addition, the tumor microenvironment is frequently hypoxic and a lack of oxygen can inhibit the activity of the fatty acid desaturase SCD1. This leads to saturation of the fatty acid pool and renders the ER less able to cope with high levels of protein synthesis, making the maintenance of adequate lipid homeostasis of paramount importance to cancer cells within hypoxic tumor domains. Tumor cells adapt to these conditions through a variety of mechanisms, including the uptake of exogenous unsaturated lipid. Both hypoxia and the presence of certain oncogenic drivers have been found to increase lipid uptake, which was found to protect cancer cells against inhibition of SCD1. Since fatty acids can be converted to triglycerides and stored within lipid droplets, we asked whether cancer cells also cope with saturation of their fatty acid pool by storing excess saturated fatty acids as triglycerides. Clear cell renal cell carcinoma (ccRCC) tumors are very fatty and lipidomic analyses have found high levels of triglycerides in ccRCC compared to normal kidney tissue. We inhibited triglyceride synthesis in A498 ccRCC cells by shRNA –mediated knockdown of DGAT enzymes, which catalyze the final step in the synthesis of triglycerides from fatty acids. We tested whether loss of DGAT activity causes sensitivity to tumor-like stresses and investigated its effect on xenograft tumor growth. Remarkably, while loss of DGAT activity has little effect on cancer cell proliferation under oxygen- and nutrient-rich conditions, loss of DGAT activity has large effects on xenograft tumor growth. We also find that loss of DGAT activity is often accompanied by increases in markers of ER stress. We are currently assessing the lipid composition of these tumors and determining what aspects of the tumor microenvironment sensitize cells to loss of DGAT activity. Citation Format: Daniel Ackerman, Bo Qiu, Hong Xie, Jurre Kamphorst, M. Celeste Simon. Assessing the role of DGAT activity on lipid homeostasis and cancer cell survival. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B33.